Ion xpress plus gdna fragment library kit

Plasmids isolated from P. tricornutum colonies transformed with plasmids containing large fragments of scaffold 25 (Supplementary Fig. 1) were transformed to E. coli and purified from agarose gels by RECO chips (Takara). Sequencing libraries were prepared for each plasmid for the Ion Torrent platform using the Ion Xpress Plus gDNA Fragment Library Kit (Life Technologies). Samples were barcoded, pooled and sequencing on an Ion Torrent 314 chip. Reads were mapped to the P. tricornutum genome using CLC Genomics Workbench and visualized using GenomeView46 (link). Plasmid p0521s was sequenced after purification using the QIAprep kit using primers Seq0521smallF, Seq0521smallR, Seq0521smallF2, Seq0521smallF3, Seq0521smallF4 and Seq0521smallR2.

Phage genomic DNA was fragmented using Ion Xpress™ Plus gDNA Fragment Library kit following the manufacturer’s protocol (Life Technologies, Foster City, CA). The fragmented DNA was collected using Pippin Prep DNA Size Selection System (Sage Science, Beverly, MA) and assessed for concentration and size distribution using a Bioanalyzer 2100 (Agilent Technologies, Mississauga, ON). The DNA fragments were then attached to the surface of Ion Sphere particles (ISPs) using an Ion Xpress Template kit (Life Technologies) according to the manufacturer’s instructions. Template-ISPs were sequenced using 316 micro-chips using an Ion Torrent Personal Genome Machine (PGM) with an Ion PGM Sequencing 400 kit (Life Technologies). The sequence reads were filtered using PGM software to remove low quality sequences, trimmed to remove adaptor sequences and the filtered sequences were assembled. The assembled genome had a coverage of 33.4×. Gaps were identified using the Lasergene® Genomics Suite of DNAStar software (DNAStar Inc., Madison, WI). The gaps were closed by PCR using primers flanking regions adjacent to the gaps and sequencing using a 3730 Genetic Analyzer (Life Technologies). The final assembled genome was manually curated for errors.